Structural basis of GPCR/G protein interactions Biochemical and biophysical approaches have identified a small number of receptor/G-alpha interaction sites using specific receptor/G-alpha combinations. However, the molecular architecture of the receptor/G-alpha interface still remains poorly defined, primarily due to the lack of studies examining receptor/G-alpha contact sites in a systematic and comprehensive fashion. However, such knowledge is essential for understanding the structural basis underlying one of the most fundamental biological processes. To address this question, we used the rat M3 muscarinic acetylcholine receptor (M3R), a prototypic Gq-coupled receptor, as a model system. We carried out systematic cross-linking experiments using Cys-substituted M3R and G-alpha -q constructs, both in the absence and presence of an activating ligand. To facilitate the interpretation of cross-linking data, we employed a modified version of the rat M3R lacking most native Cys residues (M3'(3C)-Xa). The M3'(3C)-Xa construct lacked the central portion of the third intracellular loop (i3 loop;A274K469), resulting in the removal of three Cys residues contained within this region. However, since this construct still retained the functionally critical N- and C-terminal portions of the i3 loop, it can couple to Gq-type G protein with similar efficacy as the wild-type M3R. Moreover, seven of the ten remaining endogenous M3R Cys residues were substituted with serine or alanine. A modified version of human G-alpha-q ('G-alpha-q-C-less') in which we had replaced three of the five endogenous Cys residues with alanine served as a template for Cys substitution mutagenesis. In total, we examined cross-link formation between 18 mutant M3Rs and 14 mutant G-alpha-q subunits (all constructs contained single Cys substitutions), in all possible combinations. We identified many specific M3R/G-alpha-q contact sites, both in the inactive and the active receptor conformation, allowing us to establish the first evidence-based model of the M3R/G-alpha-q interface and the nature of the conformational changes following receptor activation. Since heterotrimeric G proteins as well as most GPCRs share a high degree of structural homology, these findings should be of great general interest. Publication: Hu J, Wang Y, Zhang X, Lloyd JR, Li JH, Karpiak J, Costanzi S, Wess J. Structural basis of G protein-coupled receptor-G protein interactions. Nat. Chem. Biol. 6, 541-548, 2010. Use of yeast expression technology to identify novel M3R-interacting proteins Accumulating evidence suggests that GPCRs do not function in isolation but are part of an intricate network of receptor-protein interactions that are responsible for various functions such as trafficking and targeting of the receptor to the membrane surface, stabilization of the receptor at the cell surface, and fine-tuning of the receptors pharmacology. We recently initiated a project to identify proteins that can interact with and modulate the function of the M3R. Such proteins may represent attractive novel targets for the treatment of various pathophysiological conditions including type 2 diabetes and colon cancer. Specifically, we employed the split ubiquitin membrane-based yeast two-hybrid system (Thaminy et al., Meth Mol Biol 261, 297-312, 2004) to screen for M3R-interacting proteins. The main advantage of this system, as compared to traditional yeast two-hybrid screening approaches, is that the bait protein (the full-length M3R or any other GPCR) is localized to the plasma membrane (or other cellular membranes) and proteins that are able to interact with the bait GPCR are identified by the use of simple growth and colorimetric assays. We first prepared a yeast expression plasmid coding for a modified version of the M3R in which the C-terminus of the receptor was fused to the C-terminal portion of ubiquitin (Cub) followed by an artificial transcription factor (M3-Cub). Since the M3R is widely expressed throughout the brain, we used the M3-Cub construct as a bait to screen the NX031 human adult brain cDNA library for M3R-interacting proteins. This library coded for 'prey'proteins that are tagged with the N-terminal portion of ubiquitin (Nub). The successful interaction between bait (M3-Cub) and prey proteins (X-Nub) was assayed by monitoring yeast growth in histidine-deficient media or by using a colorimetric test for the expression of beta-galactosidase. This screen led to the identification of 68 potential M3R-interacting proteins, including proteins known to associate with the M3R such as calmodulin 2 or ARF1. The identity of the Nub-containing proteins was verified by DNA sequencing, followed by BLAST analysis. In a series of control experiments, we co-transformed all recovered Nub clones into the NMY51 yeast strain with either M3-Cub or Alg5-Cub. The co-transformants were then assayed for growth in selective media and their ability to induce color formation in a beta-galactosidase assay. This analysis confirmed that all recovered Nub clones were able to interact with the M3-Cub protein in yeast. However, none of the Nub proteins was capable of interacting with Alg5-Cub, indicative of the selectivity of the interaction of M3-Cub with the recovered Nub proteins. Interestingly, one full-length protein, transmembrane protein 147 (Tmem147;alternative name: NIFIE14;NM_032635.2), was recovered 11 independent times. Tmem147 is predicted to represent an integral membrane protein that spans the membrane six or seven times (HHMTOP or TMHMM programs). It consists of 224 amino acids and has a calculated molecular mass of 26.2 kDa. The amino acid sequence of Tmem147 is highly conserved among mammalian species (the human, rat, bovine and mouse sequences are 99% identical;see Supplemental Figure for details). The UniGene database for EST profiles indicates that Tmem147 is widely expressed throughout the body. The function of Tmem147 remains unknown at present. We are currently performing studies to examine the potential role of Tmem147 in regulating M3R expression and function.